Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2013 11(2), 505-517 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2013112-2968 eISSN: 2171-9292

Population dynamics and integrated control of the damson-hop aphid (Schrank) on hops in Spain A. Lorenzana1*, A. Hermoso-de-Mendoza2, M. V. Seco1 and P. A. Casquero1 1 Departamento de Ingeniería y Ciencias Agrarias. Universidad de León. Avda. Portugal, 41. 24071 León, Spain 2 Instituto Valenciano de Investigaciones Agrarias (IVIA). Ctra. Moncada-Náquera, km 4,5. 46113 Moncada (Valencia), Spain

Abstract The hop aphid Phorodon humuli (Schrank) (: ) is a serious pest in most areas where hops are grown. A field trial was performed on a hop yard throughout 2002, 2003 and 2004 in León (Spain) in order to analyse the population development of Phorodon humuli and its natural enemies, as well as to determine the most effective integrated program of insecticide treatments. The basic population development pattern of P. humuli was similar in the three years: the population peaked between mid to late June, and then decreased in late June/early July, rising again and reaching another peak in mid-July, after which it began to decline, rising once more in late August; this last rise is characteristic of Spain and has not been recorded in the rest of Europe. The hop aphid’s main natural enemy found on the leaves was Coccinella septempunctata (Coleoptera: Coccinellidae). The multiple regression analysis showed that aphids are positively related with the presence of beetle eggs and mean daily temperatures and negatively related with maximum daily temperature integral above 27°C in plots without insecticide treatment. The most effective program of insecticide (imidacloprid) treatments consisted of an initial treatment in June and a second treatment in the second half of July or at the beginning of August. However, a single treatment in June would be sufficient when in this last period the maximum daily temperatures were higher than 27°C for at least 15 days, avoiding in this way the harmful effects of imidacloprid on predators. Additional key words: Aphididae; Coccinelidae; Humulus lupulus; high temperature; population dynamics; con- trol; insecticide.

Introduction The hop aphid Phorodon humuli (Schrank) (Hemip- tera: Aphididae) is a serious pest in most areas where The cones or flowers of the hop (Humulus lupulus hops are grown. P. humuli can inhibit growth and L.) are of great importance to the brewing industry. reduce the number of flowers. Production losses as- This is because the lupulin they contain provides sociated with large numbers of aphids are due to reduc- beer with bitterness and other characteristic tions in the dry weight of the crop (a yield loss of 44% organoleptic or sensorial properties (Neve, 1991). was observed when the population rised to 4400 aphids The quality of bitterness is measured by the quantity m–2 in late June), rather than a reduction in alpha-acid of lupulin’s alpha-acids. At present, Spain is the content (Lorenzana, 2006). Aphid populations in hop seventh highest hop-producing country in the cones seriously reduce their economic value because European Union. Most of the plantations are situated of arbitrary commercial criteria related to the presence in the Province of León where 95% of the land of aphids in cones (Lorenzana et al., 2010), and in cultivated for hops in Spain is located (The Barth some cases can lead to total loss (Campbell, 1978; Report, 2010/2011). The most common cultivar in Thomas et al., 1983). If any aphids are noted inside León is Nugget (98%) compared with Magnum (1%) cones in Spain there is a penalty of up to 10% of the and Columbus and Perle (1%). dry weight (Lorenzana et al., 2010). The extent of the

* Corresponding author: [email protected] Received: 04-04-12. Accepted: 24-04-13 506 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517 damage caused by P. humuli has resulted in the use of Material and methods aphicides. Imidacloprid [1-(6-chloro-3-pyridyl- methyl)-N-nitroimidazolidin-2-ylidenamine], the first neonicotinoid insecticide, is particularly effective Location and methodology of sampling against sucking pests such as aphids (Zeng & Wang, 2010). As a result, most German hops are treated with Two experiments were carried out in León, Spain, this insecticide (Weichel & Nauen, 2003) and it has the first during 2002 and 2003, and the second in 2004. been widely used to control vegetable pests in other A garden planted with the hop cultivar Nugget countries, such as China (Guan et al., 2010). Imida- (0.72 ha), consisting of 40 rows (3 m apart) each with cloprid is also the most important insecticide in hop 40 plants (1.5 m apart), was chosen for the study. This cultivation in Spain (J. A. Magadán, pers. comm.). garden was situated at the University of León’s Integrated pest management encourages conser- experimental farm maintained by the School of vation of beneficial organisms while decreasing insec- Agricultural Engineering. The height of the wirework ticide use (Hoheisel & Fleischer, 2007). Coccinelids was 6 m with two strings per rootstock. Three hop bines are known to be important in regulating P. humuli po- were trained to each string. pulations on hops, as it is shown in Campbell & Cone A randomized complete block design, with five (1994) or Weissenberger et al. (1997). The integration treatments and three replicates (15 plots), was used. of natural enemies activity and imidacloprid ap- Each plot was made up of 18 plants in 3 adjacent rows plications is an efficient approach to enhancing the of 6 plants per row. The area of each plot was 81 m2 whitefly control level in Hoseini & Pourmirza (2011) (9 m × 9 m). Treatments in this study were different study. aphid densities: in 2002 and 2003 by applying the same Population development of P. humuli has been insecticide to plots at different times, and in 2004 using studied in different countries like the United States by a combination of insecticides and aphid introductions Campbell & Cone (1994), Germany by Goller et al. (Table 1). Imidacloprid was used because it was the (1997) and Benker (1997), Czech Republic by Goller standard insecticide used by Spanish growers during et al. (1997) and Zeleny et al. (1981), England by the study period. It was sprayed using a back-pack Aveling (1981), Campbell (1978) and Barber et al. sprayer in order to reduce contamination among (2003), Poland by Solarska (pers. comm.) and in France treatments. by Trouve et al. (1997), but not in a detailed way in In order to prevent the treatment of one group Spain. One way to increase selectivity of common affecting the results of neighbouring groups, in each pesticides might be to adapt them more carefully to group of eighteen plants, only the three central plants aphid population dynamics (Niehoff & Poehling, were sampled in each experimental plot. Counts were 1995), which are particularly sensitive to temperature taken in the following manner: on the surface of one change (e.g. Parry et al., 2006; Zamani et al., 2006). of the bines of the plants a wooden frame measuring Although a considerable amount of literature is 20 cm × 30 cm was randomly placed at heights of 2, available on the effect of low temperatures on aphid 3.25, and 6 m from the ground. Within the area en- mortality, there is very little information on the effects closed by this frame, counts were taken of the total of high temperatures. High temperature is a key factor number of leaves, the number of leaves with aphids, in the development of aphid populations in corn fields the total number of aphids and the average number of of the Northeastern Iberian Peninsula, playing an them per leaf attacked. The average of these counts for important role in the decrease of populations at the end each repetition was included in the statistical analysis. of June and in the relative abundance of aphid species Sampling was carried out weekly, one week measuring throughout the season (Asin & Pons, 2001). aphid population on the left bine of the plant, and the The aim of this research was to study the seasonal following week on the right. dynamics of P. humuli populations and their natural Population density of P. humuli has often been predators in hop plants. In addition, as the plants were expressed as the number of aphids per leaf, although treated for aphids at different times of year, a second other parameters can be used, such as the number of objective was to establish which program of insecticide aphids per dm2 of leaf surface (Campbell, 1978), or treatments would be most efficient in controlling the per m2 of plant surface (used by Hermoso de Mendoza aphid population in Spain. et al., 2001, for Aphis gossypii on clementines). This Phorodon humuli on hops in Spain 507

Table 1. Treatments in 2002, 2003 and 2004

Treatment Years 2002 and 2003 Treatment Year 2004 1. Untreated Without treatments 1. Untreated Without treatments and with an initial level Ipopulation of 584± 53 aphids m–2 in 25 June 2. Early Imidacloprid1 on 18 June (2002 and 2003) 2. Untreated Without treatments and with an initial level II population of 1,262 ± 131 aphids m–2 in 25 June 3. Intermediate Imidacloprid1 on 25 July (2002) 3. Untreated Without treatments and with 937 ± 88 aphids and 24 July (2003) level III m–2 in 25 June and with aphids introduced once in June and twice in July. Five leaves with aphids (150 aphids/leaf) were released on each bine at a height of between 2 and 3.25 m each time 4. Last Imidacloprid1 on 21 August (2002) and 20 August (2003) 4. Early Imidacloprid1 ground on 14 June 5. Monthly Imidacloprid1 with three repeated treatments on18 June, 25 July, 21 August (2002) and 18 June, 24 July, 20 August (2003) 5. Monthly Imidacloprid1 on14 June, 6 July and 23 August

1 0.008 L Imidacloprid /16 L water /243 m2.

study expresses aphid population density by number aphids, and unrecognised species were collected and of aphids per m2 of hop bine. Studying the number of taken back to the laboratory for identification. In 2003 aphids on the leaves within a surface area of 6 dm2 and 2004, the number of natural enemies found on from the lower, middle and upper section of the plant leaves was recorded at the same time as the P. humuli provides much more complete and representative data population was recorded. Both species name and total than counting the number of aphids on a leaf chosen number found within the frame measuring unit were at random among the plants, which is how the majority recorded. When species identification was not possible of studies have been carried out (Lorenzana, 2006). In in the field, a sample was collected for later iden- addition, it is important to emphasize that there is a tification in the laboratory. Population development of relationship between aphid population density on the most abundant natural enemies was studied, using leaves and cones (R2 = 0.895) (Lorenzana et al., 2010). the same unit as for aphids, number of natural enemies per m2 of hop bine surface area.

Population development of aphids and natural enemies Population development of aphids and temperature Sampling in 2002 began on 21st June, and terminated on 6th September. In 2003, it began on 30th May for the Maximum and mean temperatures were recorded groups ‘Untreated” and ‘Last”, whilst for the other with a local weather station located about 200 m from groups it began on 20th June, and sampling on all the hop plot in order to analyse the relationship groups finished on 29th August. The arrival of winged between them and the population development of aphids to hops during this year was also recorded, and aphids. that is why the sampling began earlier in the untreated In 2006 apterous P. humuli were reared in groups of group and in the group treated last. In 2004, it began six on plants of the Nugget cultivar in a controlled on 25th June, and terminated on 3rd September. environment room. One experiment was made at a For each year, natural enemies of P. humuli were range of constant temperatures according to the sampled on the experimental plot during the same temperature regime of the region during the hop crop period as aphid sampling. In 2002, the number of cycle in 2002, 2003 and 2004. Mean temperature was natural enemies was not recorded, although field notes around 19°C in June, 18.5°C in July and 18°C in were taken of all fauna observed whilst sampling August. The maximum temperature during these 508 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517 months exceeds 27°C some days in 2002 and 2004 and Results very frequently in 2003. The temperatures used in the experiment were 19 and 27°C. The experiment was conducted in climatic chambers with Gro-lux lights Population development of aphids and (2000 lx), 70% RH and a photoperiod of 16:8 (L:D) h. insecticide control Plants of similar age and size with two pairs of fully expanded leaves were grown in 13.5 cm diameter In broad terms it could be said that the pattern of plastic pots. Soil moisture was maintained and a P. humuli population development for the years 2002 balance liquid fertilizer was applied once a week. One (Fig. 1a) and 2004 (Fig. 2) was similar. From the first clip-cage per plant enclosing a leaf area of 3.2 cm2 was day of sampling the population showed a decrease until used. Two recently moulted adult aphids were the end of June – beginning of July, from when it then transferred to each clip-cage. They were removed 24 increased, and peaked in mid-July. It decreased again h later and their offspring reduced to six per cage. from mid-July onwards, until the end of August, when Cages were examined daily and development time was it once again began to increase. The initial quantity of recorded until the third generation. Cages were aphids in 2004 was greater than in 2002, although the carefully transferred to newly expanded leaves weekly. first drop in population was more marked in that year, and in July the peak in both years was similar. Maximum population for the year 2002 was reached Statistical analysis in the July peak, whilst in 2004 maximum population was recorded at the outset of sampling towards the end Collected data in the field were transformed using of June. the square root transformation (X + 0.5)1/2, where X are Significant differences in aphid density between the original data. This transformation is appropriate treatments for each week are shown in Fig. 1a (year for data especially with zeroes present (Steel & 2002), Fig. 1b (year 2003) and Fig. 2 (year 2004) with Torrie, 1986). These square-root values were used in capital letters. Population dynamics for each treatment the analysis of variance. Pooled analysis of variance are shown in the figures after the date of each aphicide of measurements over time appropriate to randomized application during the three years. complete block design was performed using the Statistical analysis of the different weeks studied general linear models (GLM) procedure. Analyses for during 2002 (Fig. 1a) of P. humuli population density aphid and natural enemy densities between treatments (capital letters) showed that after treatment in June, and between weeks were carried out. Mean com- the groups ‘Untreated’, ‘Last’ and ‘Intermediate’ had parisons were performed using the LSD test to examine significantly greater populations than the other groups. differences (p < 0.05) among treatments or weeks. After treatment in July, significantly greatest Linear regressions were performed between natural population was observed in the groups ‘Untreated’and enemies (beetles, lacewing eggs and mummies) per m2 ‘Last’. After the August treatment, ‘Untreated’ and of bine surface and the number of P. humuli per m2 of ‘Early’ registered the significantly greatest popula- bine surface. Stepwise multiple linear regression tions. The differences between weeks for each treat- analysis was performed in order to better understand ment in 2002 (Fig. 1a, lower-case letters) show that the the relationship between aphids and temperatures and treatments ‘Untreated’and ‘Early’had the significantly beetles. Aphid m–2 was the dependent variable, while greatest population the last week of sample (6th temperatures (mean daily temperature (MeanT), September). ‘Intermediate’ had no significant dif- maximum daily temperature integral above 27°C in the ferences in its population after the day of its treatment, fortnight before the aphid count (MaxTemInt)) that although population was greater the day after the were significant (p < 0.05) were included as indepen- aphicide treatment (26th July) and the last week of dent variables. As there were experimental units treated sample (6th September). ‘Last’ also had no significant and not treated with aphicide, the multiple regression differences in its population after the day of its treat- analysis was conducted separately by untreated and ment, although population was greater the last week treated experimental units using the PROC REG of sample (6th September). ‘Monthly’ had no sig- procedure of SAS. All analyses were performed using nificant differences in its population throughout the SAS software version 9.1.2 (SAS Institute Inc., 2004). whole sampling process. Phorodon humuli on hops in Spain 509 a) 25 35 2002 A a a

A 30 20 b

A A 25 bc bc A

1/2 c ) A

2 15 A c A A 20 cd c b A cd A 15 10 d

A Max. Temperature (0.5 + Aphids/m (0.5 + B de B c c B 10 B A ab B B e 5 ab cd cd B cd C B BC 5 B d b b ab B B a C C C B a B a b B C B C a a b b b b d d a a 0 d d a a aaa 0 21/06 28/06 05/07 12/07 19/07 26/07 02/08 09/08 16/08 23/08 30/08 06/09 Date

Untreated Early Intermediate Last Monthly Max. Temperature b) 70 40 2003 A a

60 35

30 1/2

) 50 2 b 25 40 B c a 20 Max. Temperature

(0.5 + Aphids/m 30 B a 15

20 e 10 a (Intermediate)* b (Early) 10 b (Monthly) A A 5 d d e AB d d d b e e d ab B b b B b AB e b b B b b b 0 0 30/05 06/06 13/06 20/06 27/06 04/07 11/07 18/07 25/07 01/08 08/08 15/08 22/08 29/08 Date

Untreated Early Intermediate Last Monthly Max. Temperature

Figure 1. Population development of P. humuli and maximum temperatures in the five groups of treatments (‘Untreated’, ‘Early’, ‘Intermediate’, ‘Last’ and ‘Monthly’) during the year 2002 (a) and 2003 (b). Mean comparisons between treatments (at the same date) are shown with capital letters (means followed by the same letter are not significantly different). Letters are not shown if there are no significant differences at that date. Mean comparisons between weeks (for each group) are shown with lower-case letters (means followed by the same letter are not significantly different). Population dynamics appear after the date of their treatment. Arrows (↓) indicate date of insecticide treatment (18th June for ‘Early’ and ‘Monthly’ treatments, 25th July for ‘Intermediate’ and ‘Monthly’ treatments and 21st August for ‘Last’ and ‘Monthly’ treatments in 2002, and 18th June for ‘Early’ and ‘Monthly’ treatments, 24th July for ‘July’ and ‘Monthly’ treatments and 20th August for ‘Last’ and ‘Monthly’ treatments in 2003). * Lower-case letters for ‘Intermediate’, ‘Early’ and ‘Monthly’ between 25th July and 29th August and for this reason they are not repeated between these dates. 510 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517

40 35 2002004 A a 35 30 A 30 B a A a A ab 25

1/2 B ) ab 2 C a 25 a a A b 20 A 20 B c b A B B B 15 15 dc b b b A A B b dc b Temperature Max. (0.5 + Aphids/m (0.5 + A bc BC A c BC c A de b 10 10 a d C B C C B A B C ab ab B B c c B ab ab db a c BC ab C a bc e d 5 5 a C aC de a B a AB C C a a a a bc b de de 0 bc bc a 0 25/06 02/07 09/07 16/07 23/07 30/07 06/08 13/08 20/08 27/08 03/09 Date

Untreated level I Untreated level II Untreated level III Early Monthly Max. Temperature

Figure 2. Population development of P. humuli and maximum temperatures in the five groups of treatments (‘Untreated level I’, ‘Untreated level II’, ‘Untreated level III’, ‘Early’ and ‘Monthly’) during the year 2004. Mean comparisons between treatments (at the same date) are shown with capital letters (means followed by the same letter are not significantly different). Letters are not shown if there are no significant differences at that date. Mean comparisons between weeks (for each group) are shown with lower-case letters (means followed by the same letter are not significantly different). Treatments ‘Early’and ‘Monthly’appear after the date of their treatment. Aphid sketch indicates date of introduction of aphids. Arrows (↓) indicate date of insecticide treatment (14th June for ‘Early’ and ‘Monthly’ treatments, 6th July for ‘Monthly’ treatment and 23rd August for ‘Monthly’ treatment).

Mean comparisons between treatments for the year 2002 and 2004. The peak in June was much higher and 2004 (Fig. 2, capital letters) showed that the untreated the decrease at the end of June to the beginning of July groups reached a significantly greater population than was so marked that the population practically disap- the treated groups during most weeks. If differences peared, with the following two peaks showing a very between weeks are observed (Fig. 2, lower-case letters) low aphid population in comparison to the June peak. it is remarkable that treatments without insecticides Mean comparisons between treatments for the year had the significantly greatest population the first week 2003 (Fig. 1b, capital letters) showed that on the 20th of sample (25th June), although there were no June the groups ‘Untreated’, ‘Last’and ‘Intermediate’ differences with the second week (2nd July) in the case had significantly greater populations than the other of treatment ‘Untreated level I’, and with the second groups. If differences between weeks are observed (2nd July), fifth (23rd July) and sixth (30th July) weeks (Fig. 1b, lower-case letters) it would be possible to in the case of treatment ‘Untreated level III’. ‘Early’ emphasize that treatments ‘Untreated’, ‘Early’ and treatment had the significantly greatest population the ‘Monthly’had the significantly greatest population on 23rd July, although without differences with weeks 25th 20th June. ‘Intermediate’ and ‘Last’ had no significant June to 16th July, 30th July and 13rd August. ‘Monthly’ differences in its population after the day of its treatment had no significant differences in its treatment. population during all sampling. In 2003 (Fig. 1b) there is a peak towards the end of June. A slight increase in the middle of July and a Natural enemies and aphids further slight population increase towards the end of August also occurred. In this year, the abundance of The natural enemies encountered on leaves during aphids is markedly different from those of the years sampling in 2003 and 2004 are shown in Table 2. Phorodon humuli on hops in Spain 511

Table 2. Natural enemies on hop leaves during the years 2003 and 2004

Species Abundance1 Year 2003 Coleoptera Coccinella septempunctata Linnaeus, 1758 ++++ Propylea quatuordecempunctata (Linnaeus, 1758) ++ Adalia decempunctata (Linnaeus, 1758) + Adalia bipunctata (Linnaeus, 1758) ++ Neuroptera No identified larvae + No identified eggs ++++ Thysanoptera Aeolothrips sp. + Parasitoids Mummies: no identified parasitoid +++ Year 2004 Coleoptera Coccinella septempunctata ++ Propylea quatuordecempunctata ++ Adalia decempunctata ++ Adalia bipunctata ++ Neuroptera No identified larvae + No identified eggs +++ Thysanoptera Aeolothrips sp. ++

1 +: scarce (< 5 records in 2 years); ++: regular (< 20 records in 2 years); +++: frequent (10-20 records every year); ++++: very frequent (> 20 records every year).

Coccinelids and eggs of neuroptera were the most eggs had the significantly greatest population on 2nd abundant natural enemies found on leaves during both July, although without differences with weeks 25th years. Coccinella septempunctata was the most com- June, 23rd July and 30th July in the case of beetle larvae, mon species in 2003, while this species was found in and with the week on 25th June in the case of beetle the same frequency than the rest of coccinelid species eggs. The significantly greatest population for aphids in 2004. Aeolothrips sp. was also registered in both was reached 25th June, without differences with the years, while mummies were found only in 2003. 2nd July. Statistical analysis of the different weeks Mean comparisons between weeks for beetle density studied during 2004 of beetle larvae and egg in the group ‘Untreated’ in 2003 (Fig. 3a, lower-case population density showed that after treatment in June, letters) showed that larvae and eggs had the sig- the groups without treatments had significantly nificantly greatest population on the 20th June, the greater populations than the other groups. Regression same as aphids, although without differences with the analysis showed the positive correlation between week 13rd June for the egg population. Beetle adults aphids and beetles in 2004 in the same way as 2003, had the significantly greatest population on 13rd June. although this year the greatest correlation was Statistical analysis of the different weeks studied obtained between aphids and beetle larvae (R2 = during 2003 of beetle larvae population density showed 0.8333). that after treatment in June, the groups ‘Untreated’, Population of mummies reached the significantly ‘Last’ and ‘Intermediate’ had significantly greater highest level on 20th June in 2003 (without differences populations than the rest of the groups. The group with the population during the 15th August), the same ‘Untreated’ had significantly greater adult beetle as aphids. Otherwise, lacewing eggs population did populations than the rest of the groups on 4th July. not reach the significantly greatest population at the Regression analysis showed a positive correlation same time as aphids in 2003 and 2004. Statistical between aphids and beetles in 2003 (Fig. 4). analysis of the different weeks studied during 2003 of Mean comparisons between weeks for beetle lacewing eggs population density showed that after density in the group ‘Untreated level III’in 2004 (Fig. treatment in July, the groups ‘Untreated’, and ‘Last’ 3b, lower-case letters) showed that adults, larvae and had significantly greater populations than the rest of 512 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517

a) 70 3.0 2003 A

60 b a 2.5

50 a 1/2 1/2

) B ) 2 a 2.0 2 40 C 30 b 1.5 (0.5 + Aphids/m (0.5 + bc (0.5 + Beetles/m 20 bc b c(I) c(I)* b(II) 1.0 b(II) b(II) 10 b(III) c c b b b(III) b(III) c c c c c D c b b b D b D D D D D D D 0 D D 0.5 30/05 06/05 13/06 20/06 27/06 04/07 11/07 18/07 25/07 01/08 08/08 15/08 22/08 29/08 Date Aphids/m2 Beetle Adults/m2(I) Beetle Larvae/m2(II) Beetle Eggs/m2(III)

35 3.0 b) 2004 A 30 AB a BC 2.5 a BC 25 C 1/2 1/2 ) )

2 a(III) 2 2.0 20 a(I) CD

D 15 ab ab D 1.5

(0.5 + Aphids/m D (0.5 + Beetles/m

10 b D bc b bc(I) b(III) 1.0 5 c(I) E c c(I)* b(II) b(II) b b(II) 0 b(III) b(III) b(III) 0.5 25/06 02/07 09/07 16/07 23/07 30/07 06/08 13/08 20/08 27/08 03/09 Date Aphids/m2 Beetle Adults/m2(I) Beetle Larvae/m2(II) Beetle Eggs/m2(III)

Figure 3. Population development of P. humuli and beetles (adults, larvae and eggs) in the treatment ‘Untreated’during the year 2003 (a) and in the treatment ‘Untreated level III’during the year 2004 (b). Mean comparison between weeks are shown with capital letters for aphids and with lower- case letters for beetles (means followed by the same letter are not significantly different). * Same letters between 25th July and 29th August (a) and between 6th August and 3rd September (b) and for this reason they are not repeated between these dates. Aphid sketch indicates date of introduction of aphids (b). the groups. In the case of 2004 the group ‘Untreated Temperature and aphids level III’had significantly greater populations than the rest of the groups on 16th July, 20th and 27th August. Mean generation time of P. humuli on Nugget There was no significant correlation between aphids cultivar hop plants at 19°C in the laboratory was and mummies or lacewing eggs. 10.3 ± 0.1 days. No generation was completed at 27°C, Phorodon humuli on hops in Spain 513

a) 40000 y = 6874.2x + 885.42 R 2 = 0.9122 30000 2

20000 Aphids/m 10000

0 0246 Beetle adults/m2

b) 40000 40000 y = 627.65x + 381.89 y = 595.15x + 149.84 c) 2 R = 0.9297 2 30000 30000 R = 0.9841 2 2

20000 20000 Aphids/m Aphids/m 10000 10000

0 0 0204060020406080 Beetle larvae/m2 Beetle eggs/m2 Figure 4. Correlation of the mean of the sum for each treatment between aphids and beetles in 2003: (a) y is the number of aphids per m2 and x is the number of beetle adults per m2; (b) y is the number of aphids per m2 and x is the number of beetle larvae per m2; (c) y is the number of aphids per m2 and x is the number of beetle eggs per m2. at which temperature all nymphs died from 14 ± 1.5 and in the Czech Republic (Hrdy’, 1980, quoted in days from the beginning of the experiment. Goller et al., 1997), except that the increase of population at the end of August was not found probably Temperature, natural enemies and aphids due to temperatures registered in these countries at this time. In studies carried out in England by Aveling The combination of beetle eggs, mean daily tem- (1981) in 1975, in Germany by Benker (1997) in 1996, perature (MeanT) and maximum daily temperature and in Poland by Solarska (pers. comm.) in 2004, the integral above 27°C in the fortnight before the aphid maximum peak of aphid population was also reached –2 count (MaxTemInt) explained 91% of aphid m in mid-July, with no peak in either June or August. variation in untreated plots, with beetle eggs alone Following analysis of population development over explaining 51% of the variation (Table 3). Beetle eggs 2002 in our study the conclusion is that two aphicide and MeanT had a positive regression coefficient treatments are necessary to control the population whereas MaxTemInt had a negative regression coef- during this year: the first in June and the second in ficient. No variables remained in the regression model mid-July (or beginning of August) in order to prevent in treated plots. the final population increase. The final population increase could be the most damaging if the population Discussion reaches very high levels. Regarding the population dynamics obtained in this Population development of aphids study of the year 2004 (Fig. 2), with maximum popu- lation peak at the end of June or beginning of July, the The population development for P. humuli obtained same results appear in the work carried out in England in this study will now be compared with that described by Campbell (1978) for the years 1971 and 1972 (these in other countries for hop plants where aphid pesticide plants were treated with an aphicide at the end of June) treatment was not given. Population dynamic in 2002 and in the study carried out by Barber et al. (2003) on (Fig. 1a), with the maximum peak in mid-July, coin- a series of hop plant varieties in 1997 and 1998 in the cides with that found by Campbell & Cone (1994) in same country, except that the population increase at the USA in 1992. This dynamic is also very similar to the end of August was not observed. It is worth poin- that found in Germany in 1992 by Goller et al. (1997) ting out that in the study of Barber et al. (2003) this 514 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517

Table 3. Stepwise multiple linear regression results for aphids m–2 as a function of temperatures and beetles in treated and untreated plots (2003 and 2004)

Dependent Insecticide Independent b Intercept Pr < Fa R2 variable treatment variable coefficient Aphids m–2 Untreated 12.69 Beetle eggs m–2 11.24 0.02 0.51 MeanTb 1.43 0.02 0.37 MaxTemIntc –14.79 0.05 0.03 Aphids m–2 Treated –9.45 — — — — a F: test statistic used to reject or fail to reject the null hypothesis; Pr: probability of obtaining the F test statistic, assuming that the null hypothesis is true. b MeanT: mean daily temperature. c MaxTemInt: maximum daily temperature integral above 27°C in the fortnight before the aphid count. slight population increase was observed in one of the 1992. This study shows the important role of natural varieties studied in 1998. predators, even to the point of explaining aphid The population dynamics described for 2003 population fluctuations exclusively in terms of (Fig. 1b), with a maximum population peak at the end predators. Coccinelids are probably considered the of June followed by a decrease and practical disap- main natural enemies of P. humuli worldwide, as pearance of the population for the remainder of the Tsvetkov (1962), Campbell (1973), Campbell & Cone period of cultivation, coincides with that obtained in (1994) or Weissenberger et al. (1997) emphasized in the Czech Republic by Zeleny et al. (1981) in the years their studies. Coccinelid population increased at the 1969, 1973 and 1979, in Germany by Benker (1997) same time as P. humuli population during June and in the years 1994 and 1995, in France by Trouve et al. nearly all July in Campbell & Cone (1994) study. (1997) and in England by Barber et al. (2003) in certain Adults and larvae of Neuroptera as well as parasitoids hop plant varieties for the years 1998 and 1999. In view could also be important natural enemies of aphids, of the population development over 2003 in our study although Aeolothrips sp. would probably prefer an it would be possible to conclude that the aphid alternative host. population could probably be controlled with just one In the case of this study, the first population decrease treatment in June during this year. between the end of June and beginning of July could be associated more with elevated temperatures (highest temperatures above 30°C for several days) than with Factors affecting population development natural predators, given the low number of them found. of aphids Later, temperatures fell, which produced a population peak in mid-July. The following decrease in population The multiple regression analysis performed in order could once again be attributed more to high tem- to better understand the relationship between aphids peratures than to predators, although when the and temperatures and beetles (Table 3) suggests that temperatures fell again, the population did not recover. higher concentrations of aphids are related to the Towards the end of August, the total absence of presence of beetle eggs and high mean temperatures predators and lower temperatures provided a more in untreated plots. MaxTemInt coefficient (parameter favourable habitat for aphids. estimate) is –14.79, so, for every unit increase in We believe that in the year 2003 (Fig. 1b), high MaxTemInt a 14.79 units decrease in aphids m–2. Aphid temperatures throughout the period of cultivation were population was not correlated neither with the mainly responsible for the population development temperatures nor beetles in treated plots, probably due described. In mid-June, the elevated temperatures to negative effect of imidacloprid on aphids and beetles could have provoked the abrupt population decrease, (AiZhi et al., 1999). whilst the repetition of these temperatures in the The population development obtained in the year following months prevented the population from 2002 (Fig. 1a) coincided exactly with those described recovering. It was only when the temperatures fell at by Campbell & Cone (1994) in the USA for the year the end of August that the aphid population was able Phorodon humuli on hops in Spain 515 to increase slightly. Natural enemies could have con- Lacewing eggs were found regularly with the tributed to the population decrease in mid-June, highest numbers at the end of the sample. This predator although the number of adults, larvae and eggs beetles does not seem to be efficient in controlling the hop (Fig. 3a), or lacewing eggs and mummies found was aphid, as in Zeleny et al. (1981) and Trouve (1995) studies. not very high. The beetle eggs found between the Following the analysis of those factors which could beginning and middle of June were the source of the have affected population development of P. humuli larvae peak a week later. Aphid numbers continued to throughout the three years of sampling, it is possible fall after predator numbers peaked because the daily to conclude that in 2002 and 2004, the temperature had consumption rate of larvae for hop aphids is positively a certain effect, although the influence of natural correlated with larval instar (Campbell & Cone, 1994). enemies should also be taken into account. High The beetle eggs found in the latter half of June were temperature was not the primary reason soybean aphid the source of the larvae found at the end of this month, populations remain low in Missouri (USA), as some although their number was very low probably due to speculate, being more likely that resident predators are temperatures higher than 30°C. responsible (Meihls et al., 2010). In 2003, high tem- In 2004, the population of aphids fell between the peratures meant that in a very short time the population end of June and beginning of July, the same as in the practically disappeared. Temperature is one of the main two previous years (Fig. 2). The population of adults, factors that affect development rate, fecundity and larvae and eggs beetles (Fig. 3b) reached its maximum lifespan of aphids (Aalbersberg et al., 1987). The peak at the same time as aphids in the majority of optimum constant temperature for P. hunuli is near groups, just before this fall. Peak abundance of 18°C-20°C and suffers from heat stress as temperature coccinelids coincided with the peak of P. humuli in July increase (C. A. M. Campbell, pers. comm.). Campbell in the study of Campbell & Cone (1994). Besides the (1983) reared aphids on plants of the cvs. Tolhurst, influence of natural enemies, aphids in the present Fuggle and Northern Brewer at different temperatures study could have survived high temperatures for an in a controlled environment room. Mean generation extended time, which would have contributed to the times at temperatures fluctuating between 15° and fall in aphid population. Beetle larvae numbers also 20°C (10.7 ± 0.1, 10.4 ± 0.1 and 10.5 ± 0.1 days res- declined, but the daily consumption rate is positively pectively in the three varieties) were similar to those correlated with instar (Campbell & Cone, 1994). found in cv. Nugget in this study at 19 (10.3 ± 0.1 Following a slight drop in temperature, degrees rose days). Development was faster at 25°C than at 20°C, again between the beginning and middle of July, and although size, net fecundity and life expectancy were the population began to recover slightly from this date reduced. Thus Campbell (1983) established that 25°C onwards. This fact leads to the conclusion that it is not may be near the upper thermal threshold for P. humuli. only temperature which affects aphid population Thanks to the results obtained in this study we can development. Larvae completed their larval develop- establish an upper thermal threshold of less than 27°C ment and pupated, allowing the aphid population to for this aphid. Although are not subjected to recover. Later high temperatures could have caused the constant temperatures in nature, controlled laboratory mid-July peak to be minor, as well as the presence of studies can provide a valuable insight into the popu- some natural enemies observed at this time in some of lation dynamics of aphids (Satar et al., 2005). In this the treatments. The peak which occurred in the group way, the results obtained in our experiment confirm ‘Untreated level III’ was much more marked than in the explanation of the phases of the hop aphid po- any of the other groups due to aphids having been in- pulation dynamics in Spain. The upper optimal troduced twice. This peak coincided with a new increa- temperature for Acyrthosiphon pisum on pea was se in the number of larvae. Aphid population decreased 23.1°C in Morgan et al. (2001) study, where the highest later, as well as larvae population, although the final temperature used (26.7°C) had a deleterious effect on instars larvae eat many more aphids than the first ones. aphid development. High temperature is a key factor Aphid population recovered slightly when these larvae pu- in the development of aphid populations in corn fields pated. Population development went on to display the of Northeastern Iberian Peninsula, where the mortality same pattern as in the previous two years, and the slight of Sitobion avenae (Fabricius) nymphs reached 100% rise in population which occurred at the end of August at 30°C and that of Metopolophium dirhodum (Walker) was explained in the same way as for the year 2002. reached 100% at 27.5°C (Asin & Pons, 2001). 516 A. Lorenzana et al. / Span J Agric Res (2013) 11(2), 505-517

In conclusion, it is important to emphasize the basic and their effects on their predators. J Henan Agric Sci population development pattern of P. humuli: the 4: 25-26. population peaked between mid to late June, and then Asin L, Pons X, 2001. Effect of high temperature on the growth and reproduction of corn aphids (Homoptera: decreased in late June/early July, rising again and Aphididae) and implication for their population dynamics reaching another peak in mid-July, after which it began on the Northeastern Iberian Peninsula. Environ Entomol to decline, rising once more in late August. This 30: 1128-1137. increase in the population of aphids in August is Aveling C, 1981. The role of Anthocoris species (Hemip- characteristic of Spain and was different from the tera: Anthocoridae) in the integrated control of the studies reported in the rest of Europe. High tem- damson-hop aphid (Phorodon humuli). Ann Appl Biol 97: 143-153. peratures had a negative effect on the aphid population. Barber A, Campbell CAM, Crane H, Darby P, Lilley R, 2003. In addition, it would be necessary to take into account Costs-benefits of reduced aphicide usage on dwarf hops the influence of natural enemies on the aphid popu- susceptible and partially resistant to damson-hop aphid. lation. Following analysis of population development Ann Appl Biol 143: 35-44. over three years of study, it was concluded that most Benker U, 1997. Population dynamics of pests (damson-hop effective treatments with aphicides were: an initial aphid Phorodon humuli (Schrank) and two-spotted spider mite Tetranychus urticae Koch) and beneficial organisms treatment in June, when natural predators have not yet in hops. Proc Sci Commiss Int Hop Growers’Convention, v appeared on the hop plants, and a second treatment in Zatec (Czech Republic), Aug 3-6. p: 5. mid-July (or beginning of August) in order to prevent Campbell CAM, 1973. Studies on the ecology of the the final population increase, although with this damson-hop aphid [Phorodon humuli (Schrank.)]. treatment, any possible beetles which might have Doctoral thesis. London Univ, UK. reappeared at the end of August would be eliminated. Campbell CAM, 1978. Regulation of the damson-hop aphid [Phorodon humuli (Schrank.)] on hops (Humulus lupulus When in mid-July (or beginning of August) tem- L.) by predators. J Hortic Sci 53: 235-242. peratures are higher than 27°C for at least 15 days, a Campbell CAM, 1983. Antibiosis in hop (Humulus lupulus) single treatment applied in June would be sufficient, to the damson-hop aphid, Phorodon humuli. Entomol Exp avoiding in this way the harmful effects of imidacloprid Appl 33: 57-62. on predators. Campbell CAM, Cone WW, 1994. Influence of predators on population development of Phorodon humuli (Homoptera: Aphididae) on hops. Environ Entomol 23: 1391-1396. Goller E, Nunnenmacher L, Goldbach HE, 1997. Faba beans Acknowledgments as a cover crop in organically grown hops: influence on aphids and aphid antagonists. Entomol Res Organ Agr 15: We thank Colin Campbell, Florian Weihrauch, 279-284. David James and José Antonio Magadán for providing Guan H, Chi D, Yu J, Li H, 2010. Dynamics of residues from information, and we thank Piedad Campelo, Blanca a novel nano-imidacloprid formulation in soybean fields. Crop Prot 29: 942-946. Ramírez, Nicolás Pérez, Alfonso Pérez and Juan Luis Hermoso de Mendoza A, Belliure B, Carbonell EA, Real V, Gutiérrez for their help in the field and laboratory 2001. Economic thresholds for Aphis gossypii (Hemiptera: work. We also thank the Junta de Castilla y León Aphididae) on Citrus clementina. J Econ Entomol 94: (Spain) for the support of the work (project N°. 439-444. LE57/O2) and the Fundación Chicarro-Canseco- Hoheisel GA, Fleischer SJ, 2007. Coccinellids, aphids, and Banciella for the grant awarded to A. L. inside the pollen in diversified vegetable fields with transgenic and isoline cultivars. J Ins Sci 7: 61. Available in: http://insect- University of León. science.org/7.61/ [27 March, 2012]. Hoseini SA, Pourmirza AA, 2011. Short communication. Evaluation of the efficiency of imidacloprid and Encarsia References inaron Walker (Hymenoptera:Aphelinidae) integration to control the whitefly, Trialeurodes vaporariorum West- Aalbersberg YK, Du Toit F, Van Der Westhuizen MC, Hewitt wood (Homoptera: Aleyrodidae), under greenhouse con- PLl, 1987. Development rate, fecundity and lifespan of ditions. Span J Agric Res 9(3): 906-911. apterae of the Russian wheat aphid, Diuraphis noxia Lorenzana A, 2006. Determinación de los umbrales de (Mordvilko), Hemiptera: Aphididae, under controlled tratamiento del pulgón del lúpulo Phorodon humuli conditions. Bull Entomol Res 77: 629-355. (Schrank, 1801) y estudio de la evolución poblacional en AiZhi L, ShiGong L, SuHua H, 1999. The effectiveness la provincia de León. Doctoral thesis. León Univ, Spain. of imidacloprid and pirimicarb against wheat aphids [In Spanish]. Phorodon humuli on hops in Spain 517

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